Enzymes are protein biomolecules that are able to function as highly effective, high-performing biological catalysts and are fundamental for all biological life. They are substances that accelerate the chemical reactions of life without being consumed themselves. Isolated enzymes are important in many industrial processes for treating biological substrates. Examples of industries that benefit from the use of enzymes are food and feed industry, detergent industry, leather industry and the increasing application of enzymes in bioenergy industry as exemplified by the production of bioethanol. All the above industries employ various hydrolytic enzymes such as amylases, cellulases, proteases, lipases etc. for increasing production rate and yield of products from biobased feed stock[1]. There are numerous other conceivable applications in related fields e.g. for production of biogas or break down of harmful compounds in process water. Thus, enzymes for industrial and environmental applications have a large and increasing economic and ecological value.
One bottleneck in the application of enzymes in industrial processes is that in order to be active, enzymes and other proteins must keep a highly ordered structure. However, the highly ordered structure of proteins is only maintained if the proteins are stable at the prevailing conditions, i.e. pH, ionic strength, temperature, etc., within certain limits that are specific for each type of protein. In terms of natural selection of proteins during evolution, this notion stresses the fact that a protein molecule only makes structural sense when it exists under conditions similar to those for which it was selected, or the so called, native state. That is, the enzymes for treating biological substrates should ideally be evolutionarily adapted to the environment in which they are to be used, to assure that the enzymes have a high activity and longevity in that unique environment. If however the protein is not stable enough for the application in mind the stability of the enzyme needs to be altered by various protein engineering methods[2], which is a very complex and expensive process with an often uncertain outcome.
Microorganisms are a valuable source of industrially important enzymes. Microorganisms are the smallest form of life, nevertheless they collectively constitute the largest mass of living material on earth. Not only are they the most abundant, but the diversity of microbial life by far exceeds that of the plants and animals, which is a result of microorganisms' ability to live at places unsuitable for other organisms. An ability that comes from microorganisms' highly specialized physiological capabilities. Part of these capabilities comes from having enzymes that are stable, active and adapted to different habitats so that different microorganisms can feed and make use of the nutrients available in different environments.
To feed and utilize nutrition in their environment, microorganisms are generally not able to break down more complex compounds like polysaccharides and proteins within the cell. Their only way to digest them is via extracellular enzymes. For example, to break down starch into glucose the cells secrete amylases into their environment in what could be described as an external digestion process. In the environment the amylases break down starch until the microbes are surrounded by small glucose molecules which can then easily be ingested.
Microorganisms can secrete both cell-bound and free extracellular enzymes. Cell-bound enzymes are important for the microorganisms since they exert their activities close to the microbial cell, which assures that e.g. the glucose produced from the starch can be ingested by the very organism that produced the amylases. For industrial applications however extracellular free enzymes are of larger interest than cell-bound enzymes as they are adapted to be stable independently of the cell and furthermore since the efficiency in degrading complex compounds is far higher for an excreted free enzyme than for a cell-bound one. All microorganisms, both prokaryotic and eukaryotic, have the ability to produce extracellular enzymes. However, for the large scale production of enzymes from isolated genes, prokaryotic enzymes are most often preferred as they lack posttranslational modifications and are thereby easier and cheaper to produce in heterologous expression systems.
Thus, as described above, the large diversity of microorganisms that has evolved in different environments offers an almost inexhaustible bio-bank of organisms with the ability to produce enzymes with different functions in different environments, suitable for various industrial processes. In nature, microorganisms live in association with other microorganisms in populations. The environment in which a population lives is called a habitat in which different populations interact in assemblages called microbial communities. The diversity and abundance of microorganisms in a microbial community is dependent on the resources, i.e. food, and conditions such as temperature, pH, oxygen content etc., that exist in that environment. Microbial habitats can be found in all environments that can sustain life. These includes familiar oxic and anoxic habitats like soil, water, animals and plants but also extreme environments with high or low pH, high or low temperature, high pressure, high salt concentration etc. Following the above reasoning, microorganisms that have evolved to live in any of the above habitats naturally also secrete enzymes that are stable in that very environment.
Microbial populations in a community interact and cooperate in various ways, some of which are beneficial to the whole community. For example, the waste products of metabolic activities of some microorganisms can be nutrients for others. A special case of interaction is syntrophy (literarily meaning “eating together”), a situation in which two or more microorganisms team up to degrade a substance that neither microorganism can degrade individually.
Due to technical limitations, historically and traditionally, enzymes have been discovered and isolated from pure cultures of microorganisms. The process of obtaining a pure culture of a microorganism is very time consuming, if at all possible. A microorganism of course needs to be known to produce the product or enzyme of interest, preferably at the conditions of interest. This microorganism then needs to be purified from all the other microorganisms in its habitat by differential and selective media in hope to get the microorganism in a pure culture. However, recent findings by metagenomics, where the whole microbial community is considered to represent a meta-organism, have made it clear that the interactions and co-operations between populations of microorganism in a microbial community are so vital that only a fraction (1-10%) of all microorganisms can be obtained in pure cultures. Consequently, the enzymes that have been found so far and are in use in industrial applications today are very much biased towards gene products of the very few microorganisms that can be obtained in pure cultures. Thus, there are some 90-99% of the microorganisms that are not accessible for screening for valuable enzymes with methods that rely on pure cultures of microorganisms[3].
In view of the limitations of pure culturing described above, other methods to enable screening for novel enzymes in full microbial communities are desirable. One way of doing this is to take the route via DNA, using metagenomics and produce a meta-genomic library of microbial communities in natural habitats[4]. However, one drawback of this approach is that all genes need to be cloned, even those that might not be of interest for industrial biotechnology, such as genes coding for e.g. structural proteins. Another drawback of this approach is that the huge number of clones that are produced, all carrying different gene segments, have to be expressed and submitted to either sequence or activity based screening for identification of the correct enzyme.
Recent progress in proteomics, i.e. the large-scale study of proteins expressed by an organism, has also lead to the development of metaproteomics, i.e. the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time[5]. However, up till now metaproteomics has only been used to collect intracellular proteins in order to understand metabolic pathways and interactions among the populations that makes up a certain microbial community[6-8]. Even if it would be desirable to screen for secreted proteins/enzymes in environmental samples this would not be possible since the sample would be too contaminated with interfering substances and the enzymes would be too diluted. For the purpose of screening for secreted proteins (known as secretomics) by microorganisms, so far only microorganisms in pure cultures have been used, often with the aim of finding virulence factors for pathogenic microorganisms[9, 10].
The growing worldwide interest to increase the production of biogas from organic residues, to be used as an alternative fuel, can serve as an important example of an industrial process that clearly would benefit from employing hydrolytic enzymes. However, this will only be possible if enzymes that are effective and have a long lifetime in the prevailing conditions of an anaerobic digester can be found. Much of the organic matter used as substrate in biogas production has a low biological availability, due to being physically and chemically stable, which results in that some substrates display a low degree of degradation. Most suggested pretreatment methods for increasing biogas production rely on energy intensive thermal and/or physical disintegration of various cell walls, with the intention to release the content of the cells. Examples are steam explosion, ultrasonication, electroporation, bead-milling etc. However, even if successful and economically viable, these methods alone will only marginally influence the rate and degree of degradation of the actual cell walls and other structural components that make up a significant part of most organic material.
Although established in many other industrial biotechnological applications, the addition of hydrolytic enzymes in order to increase both the rate and yield of digestion in the biogas production is fairly new. The use of enzymes for this purpose is applicable in processes where the first and second step in methanogenesis, i.e. disintegration and hydrolysis, are rate limiting (see FIG. 1). The idea is that enzymes with specific activity towards various biopolymers such as proteins and polysaccharides will hydrolyze the organic matter and that the addition of hydrolytic enzymes to the process leads to a more effective use of substrates that are difficult to degrade, and to the possible use of various new substrates. Experiments have been made by adding various commercially available hydrolytic enzymes to anaerobic digesters. However, these enzymes have originated from various microorganisms that are not part of microbial communities in methanogenic habitats. Thus, those enzymes are not evolutionarily adapted to the prevailing conditions in an anaerobic digester and the intended use. Consequently, experiments made so far have generally not shown any high success rate.
However, what is clear is that upon addition of recalcitrant organic material, such as cellulose, to an anaerobic digester there is an increase in gas production although this process is very slow. Thus, within the microbial community there are microorganisms present that are able to synthesize and secrete enzymes that are active against e.g. cellulose at the prevailing conditions, but in too small amounts. Such an enzyme or enzymes are of course of a considerable value in increasing biogas production if it would be possible to identify, produce and add them to anaerobic digesters.